Project Summary/Abstract Glaucoma is a major cause of blindness and current treatments are insufficient. A major and the only treatable risk factor for glaucoma is elevated intraocular pressure (IOP), which is usually due to trabecular meshwork (TM) dysfunction. Remarkably, techniques for directly assessing the most relevant measure of TM function, i.e. outflow facility, have not changed for 60+ years, are patient-unfriendly, and are rarely used clinically. Our prior work has established a correlation between TM stiffness and outflow facility in human and mouse eyes, strongly implicating TM stiffness as a surrogate measure of TM function. Here it is proposed to develop and validate a novel OCT-based method to measure TM stiffness in patients as an indirect indicator of TM function, an approach we term ?21st century tonography?. The key idea is to image the TM and Schlemm?s canal as IOP is manipulated. Based on these images, unbiased (automated) OCT image segmentation will be used to quantify the change of Schlemm?s canal luminal size as a function of IOP, and then engineering analysis techniques (inverse finite element modeling) will be employed to quantify the stiffness of the TM in the living eye. The proposal?s preliminary data strongly suggest that this approach is feasible. Thus, the overall objective is to validate the approach, which will be achieved through two specific aims. The first uses mouse models and the second uses human eyes. In Aim 1, we will build on our extensive experience in imaging the mouse outflow tract with OCT in normotensive animals and in two clinically-relevant established models of ocular hypertension. The resulting TM stiffness measurements will be validated against direct measurements of TM stiffness using our established protocol based on atomic force microscopy, and against longitudinal IOP and outflow facility measurements. In Aim 2, we will carry out analogous studies in human eyes, first using perfused human anterior segments where the tissue can be extensively manipulated, and then moving to clinical studies in patients with ocular hypertension/early glaucoma. An important aspect of all proposed studies is that the effects of a clinically- available rho-kinase inhibitor (netarsudil) on TM stiffness, TM function, and IOP will be longitudinally assessed, strengthening clinical relevant and impact. It is expected, as suggested by the strong preliminary data, that the proposed OCT-based approach to measuring TM stiffness and TM function will be shown to be valid. This project is highly innovative, since it will create a novel, non-invasive tool to interrogate TM function in human subjects, the first such tool since the introduction of tonography six decades ago. Such a tool will be useful in multiple contexts, including: (1) basic science studies of TM function and physiology; and (2) longitudinal evaluation of novel emerging treatments to repair TM function, including small molecule-based therapies, gene therapy approaches for restoring TM function, and stem cell-based therapies for the TM.